MECHANISMS OF EPIDERMAL HOMEOSTASIS AND EARLY NEOPLASIA PROJECT SUMMARY Changes in cell metabolites and Ras signaling control epidermal homeostasis and neoplasia, however, the regulators involved are incompletely defined. Using large-scale metabolomic analyses during epidermal differentiation, AR43799 uncovered an essential role for accumulation of free intracellular glucose. These data support a model in which a specific glucose importer, SGLT1, and a glucose-binding transcription factor, IRF6, anchor a pro-differentiation positive feedback loop in epidermis that is disrupted in early neoplasia. In parallel efforts, proximity proteomics followed by knockouts identified new protein transporters essential for the trafficking and function of KRAS, HRAS, and NRAS. These transporters included SNARE and non-SNARE proteins; among the latter is LZTR1, a Golgi-localized protein mutated in the skin-affecting RASopathy, Noonan syndrome. This AR43799 competing renewal will define the function of these new glucose mediators and Ras transporters in both homeostasis and early neoplastic progression in the epidermis. First, we will characterize candidate glucose effectors to define the role of intracellular glucose accumulation in epidermal homeostasis and neoplasia. The postulated glucose-enabled positive differentiation feedback loop centered on SGLT1 and IRF6 will be disrupted in epidermal tissue and impacts on homeostasis quantified. Based on the finding that epidermal cancers suppress SGLT1 and IRF6 levels, that both are required for differentiation gene induction, and that Ras suppresses glucose levels in normal cells, we will also define the impacts of perturbing these glucose-linked regulators, as well as glucose homeostasis itself, on early Ras-driven neoplastic progression in epidermis. Aim I will test the hypothesis that a glucose-regulated SGLT1-IRF6 positive feedback mechanism enables epidermal differentiation and opposes early neoplastic progression. Second, we will characterize new transporters in Ras-mediated control of epidermal homeostasis and neoplasia. Preliminary studies identified SNARE transporters that specifically deliver KRAS to its plasma membrane site of signaling. To define the full set of transporters required for each Ras isoform, the impact of deleting each putative transporter on individual Ras isoform trafficking and signaling will be assessed in normal keratinocytes. The impact of each transporter on Ras-isoform enabled epidermal tissue homeostasis and, separately, early epidermal neoplasia, will also be examined in vivo. Aim II will test the hypothesis that Ras isoform-specific transporters, are essential for Ras action in epidermal homeostasis and neoplastic progression. At the end of proposed funding, this effort will define the function of newly identified glucose regulators and Ras transporters in epidermal homeostasis and early neoplasia.